1- Making Density Gradients

1. 1- Making Density Gradients Pre-formed discontinuous gradients Pre-formed continuous gradients Self-generated gradients Training File 2 topicsTraining File 2 topics

2. Discontinuous gradient by overlayering If we make up, for example 3 aqueous solutions of, for example, iodixanol (10%, 20% and 30%, w/v) we can make a discontinuous gradient by overlayering; each solution (starting with the densest) is overlayered with successively lighter solutions from either a pipette or syringe attached to a wide-bore flat-tipped metal tube (cannula). Overlayering is best accomplished if the tube is tilted (as in C) and the tip of the pipette or syringe placed approx 0.5 cm above the meniscus.If we make up, for example 3 aqueous solutions of, for example, iodixanol (10%, 20% and 30%, w/v) we can make a discontinuous gradient by overlayering; each solution (starting with the densest) is overlayered with successively lighter solutions from either a pipette or syringe attached to a wide-bore flat-tipped metal tube (cannula). Overlayering is best accomplished if the tube is tilted (as in C) and the tip of the pipette or syringe placed approx 0.5 cm above the meniscus.

3. Discontinuous gradient by underlayering The less commonly used method of underlayering (low density end first) is in fact the more satisfactory method. It is only easily accomplished using a syringe and metal cannula and the hemispherical section of the bottom of the tube aids smooth flow. After adding the least dense solution, the next most dense solution is taken into the syringe – if for example you need to layer 3 ml, then take up approx 5 ml and expel the liquid to the 4 ml mark (this ensures there are no air bubbles in the cannula. Wipe the outside of the cannula with a tissue to remove any excess liquid and introduce the tip of the cannula to the bottom of the tube, moving it smoothly down the wall of the tube. Smoothly introduce 3 ml of the solution. Depressing the syringe plunger to the 1ml mark rather than to the bottom of the barrel is both more accurate and ensures any air bubbles, trapped on the plunger, do not enter the tube. After a few seconds withdraw the syringe, again keeping the tip of the cannula against the wall of the tube and repeat the procedure. The less commonly used method of underlayering (low density end first) is in fact the more satisfactory method. It is only easily accomplished using a syringe and metal cannula and the hemispherical section of the bottom of the tube aids smooth flow. After adding the least dense solution, the next most dense solution is taken into the syringe – if for example you need to layer 3 ml, then take up approx 5 ml and expel the liquid to the 4 ml mark (this ensures there are no air bubbles in the cannula. Wipe the outside of the cannula with a tissue to remove any excess liquid and introduce the tip of the cannula to the bottom of the tube, moving it smoothly down the wall of the tube. Smoothly introduce 3 ml of the solution. Depressing the syringe plunger to the 1ml mark rather than to the bottom of the barrel is both more accurate and ensures any air bubbles, trapped on the plunger, do not enter the tube. After a few seconds withdraw the syringe, again keeping the tip of the cannula against the wall of the tube and repeat the procedure.

4. Diffusion of a discontinuous gradient If a discontinuous gradient of say 3 ml each of 10%, 20%, 30% and 40% iodixanol in a 14 ml tube is allowed to stand for several hours at room temperature or overnight at 4°C, then the sharp discontinuities of the stepped density profile become smoothed out by diffusion of the solute across each interface and eventually the gradient will become smooth and continuous. This is a commonly used strategy to make a continuous gradient. A linear gradient will be produced if the density increment between each step is the same and the volume of each step is the same. If either the volume of one or more of the steps is changed or the density interval of each step is changed the shape of the gradient can be made concave or convex or irregular in order to accentuate the separation of particular particles. If the diffusion time and temperature are well controlled the gradient shapes are highly reproducible.If a discontinuous gradient of say 3 ml each of 10%, 20%, 30% and 40% iodixanol in a 14 ml tube is allowed to stand for several hours at room temperature or overnight at 4°C, then the sharp discontinuities of the stepped density profile become smoothed out by diffusion of the solute across each interface and eventually the gradient will become smooth and continuous. This is a commonly used strategy to make a continuous gradient. A linear gradient will be produced if the density increment between each step is the same and the volume of each step is the same. If either the volume of one or more of the steps is changed or the density interval of each step is changed the shape of the gradient can be made concave or convex or irregular in order to accentuate the separation of particular particles. If the diffusion time and temperature are well controlled the gradient shapes are highly reproducible.

5. Rapid formation of continuous from discontinuous gradient If the tube containing the discontinuous gradient is capped and very carefully and smoothly rotated to a horizontal position, the interfacial area increases and the distance between each interface decreases, so diffusion and formation of a continuous gradient will occur much more rapidly. At room temperature, a continuous gradient of iodixanol in a 14 ml tube can be formed in approx 45 min. This should not be attempted the morning after the night before.If the tube containing the discontinuous gradient is capped and very carefully and smoothly rotated to a horizontal position, the interfacial area increases and the distance between each interface decreases, so diffusion and formation of a continuous gradient will occur much more rapidly. At room temperature, a continuous gradient of iodixanol in a 14 ml tube can be formed in approx 45 min. This should not be attempted the morning after the night before.

6. Two-chamber device for continuous gradients Two identical chambers (A and B) connected by a tapped channel (T) contain equal volumes of the high density and low density solutions respectively and identical magnetic stirring bars (SB). Chamber B, which sits on a magnetic stirrer (M), bears a delivery tube which reaches to the bottom of a centrifuge tube via a low-pulsation peristaltic pump (P). When the levels of liquid in the two chambers fall synchronously, dense solution from A mixes continually with the lighter solution in B so that the pump delivers a solution of ever-increasing density to the bottom of the centrifuge tube. The two stirring bars ensure that the level of liquid in A and B is the same. If the two solutions have very different densities and if the stirring is not sufficiently vigorous, the dense solution may flow under the lighter solution. If the placement of the two solutions is reversed, then the tendency of the less dense solution two float up through the denser solution in B will improve mixing considerably. In this format the tip of the delivery tube needs to be placed against the wall of the tube, near its top, so that solution of decreasing density flows to the bottom. If the flow down the wall of the tube is not continuous (but in the form of drops) this can also cause mixing. Use of the Labconco Auto Densi-flow machine (see Graphic 31) to deliver the gradient into the tube avoids this problem totally in the dense-end first mode. A single two-chamber gradient maker can be used to create multiple gradients but any manifold in the delivery line must be situated before P.Two identical chambers (A and B) connected by a tapped channel (T) contain equal volumes of the high density and low density solutions respectively and identical magnetic stirring bars (SB). Chamber B, which sits on a magnetic stirrer (M), bears a delivery tube which reaches to the bottom of a centrifuge tube via a low-pulsation peristaltic pump (P). When the levels of liquid in the two chambers fall synchronously, dense solution from A mixes continually with the lighter solution in B so that the pump delivers a solution of ever-increasing density to the bottom of the centrifuge tube. The two stirring bars ensure that the level of liquid in A and B is the same. If the two solutions have very different densities and if the stirring is not sufficiently vigorous, the dense solution may flow under the lighter solution. If the placement of the two solutions is reversed, then the tendency of the less dense solution two float up through the denser solution in B will improve mixing considerably. In this format the tip of the delivery tube needs to be placed against the wall of the tube, near its top, so that solution of decreasing density flows to the bottom. If the flow down the wall of the tube is not continuous (but in the form of drops) this can also cause mixing. Use of the Labconco Auto Densi-flow machine (see Graphic 31) to deliver the gradient into the tube avoids this problem totally in the dense-end first mode. A single two-chamber gradient maker can be used to create multiple gradients but any manifold in the delivery line must be situated before P.

7. The Gradient Master The Gradient Master (and its less expensive small brother, the Gradient Mate) are manufactured by Biocomp (Fredericton, New Brunswick, Canada). The two solutions (e.g. 10% and 40% iodixanol) are layered in a tube which is capped and placed in the vertical barrel (B) which accommodates up to 6 tubes (depending on the tube volume). The barrel then tilts automatically (usually to 80° to the vertical) and rotates at approx 20 rpm for 2-3 min. The tilting increases the interfacial area between the two solutions and the gradient is formed by the controlled mixing. The barrel returns to a vertical position at the end of the mixing period. The viscosity of the two solutions and the shape of the desired gradient influence the required values for the time and rpm parameters. It is a particularly good device to use if the sample is to be included in one or both of the layers (see Training File 1 for sample/gradient handling formats).The Gradient Master (and its less expensive small brother, the Gradient Mate) are manufactured by Biocomp (Fredericton, New Brunswick, Canada). The two solutions (e.g. 10% and 40% iodixanol) are layered in a tube which is capped and placed in the vertical barrel (B) which accommodates up to 6 tubes (depending on the tube volume). The barrel then tilts automatically (usually to 80° to the vertical) and rotates at approx 20 rpm for 2-3 min. The tilting increases the interfacial area between the two solutions and the gradient is formed by the controlled mixing. The barrel returns to a vertical position at the end of the mixing period. The viscosity of the two solutions and the shape of the desired gradient influence the required values for the time and rpm parameters. It is a particularly good device to use if the sample is to be included in one or both of the layers (see Training File 1 for sample/gradient handling formats).

8. Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time Increasing the time of rotation generally makes the gradient more linear and more shallowIncreasing the time of rotation generally makes the gradient more linear and more shallow

9. Swinging-bucket rotor There are two traditional types of rotor: swinging-bucket and fixed-angle. The swinging-bucket rotor is by far the most widely used for density gradient centrifugation. In the swinging-bucket rotor, at rest, the tube and bucket are vertical and the meniscus of the liquid is at 90° to the earth’s vertical centrifugal field. During acceleration of the rotor the bucket, tube and meniscus reorient through 90° in the spinning rotor’s radial centrifugal field. Any gradient in the tube reorients with the tube so that interfaces are also always perpendicular to the centrifugal field.There are two traditional types of rotor: swinging-bucket and fixed-angle. The swinging-bucket rotor is by far the most widely used for density gradient centrifugation. In the swinging-bucket rotor, at rest, the tube and bucket are vertical and the meniscus of the liquid is at 90° to the earth’s vertical centrifugal field. During acceleration of the rotor the bucket, tube and meniscus reorient through 90° in the spinning rotor’s radial centrifugal field. Any gradient in the tube reorients with the tube so that interfaces are also always perpendicular to the centrifugal field.

10. Fixed-angle and vertical rotors The other two types of rotor are fixed-angle and vertical. A fixed angle rotor is normally used for simple pelleting of particles but there are examples in which these rotors are used for gradient work. A vertical rotor is the most efficient way of doing density gradient separations of membranes, viruses and macromolecules (see next 11 and 12). The tube is maintained in vertical position and the gradient reorients in the tube, illustrated with a discontinuous gradient in the graphic. A gradient will reorient in the same way in a fixed-angle rotor tube. The acceleration and deceleration between 0 and 2000 rpm, during which the reorientation occurs must be achieved slowly and smoothly to avoid disturbance to the gradient.The other two types of rotor are fixed-angle and vertical. A fixed angle rotor is normally used for simple pelleting of particles but there are examples in which these rotors are used for gradient work. A vertical rotor is the most efficient way of doing density gradient separations of membranes, viruses and macromolecules (see next 11 and 12). The tube is maintained in vertical position and the gradient reorients in the tube, illustrated with a discontinuous gradient in the graphic. A gradient will reorient in the same way in a fixed-angle rotor tube. The acceleration and deceleration between 0 and 2000 rpm, during which the reorientation occurs must be achieved slowly and smoothly to avoid disturbance to the gradient.

11. Sedimentation path length of rotors In a swinging-bucket rotor the sedimentation path length is the length of the tube. In a vertical rotor the sedimentation path length is the diameter of the tube. The vertical rotor is therefore the most efficient rotor; at the same RCF, particles will reach their banding density much more quickly in a vertical rotor than in a swinging-bucket rotor. The fixed-angle rotor would occupy an intermediate position. But in a vertical rotor sedimenting particles cannot encounter the wall of the tube in the same way as they do in a fixed-angle rotor. In addition, for tubes of the same volume and dimensions (rotating at the same rpm), the hydrostatic pressure experienced by a particle is much less in a vertical rotor than in a swinging-bucket rotor, since the height of the liquid column is much smaller. The hydrostatic pressure is a function of the square of the height of the liquid column and this pressure has been shown to damage some organelles. In short, for density gradient centrifugation, the vertical rotor has none of the disadvantages of either the swinging-bucket rotor or the fixed-angle rotor.In a swinging-bucket rotor the sedimentation path length is the length of the tube. In a vertical rotor the sedimentation path length is the diameter of the tube. The vertical rotor is therefore the most efficient rotor; at the same RCF, particles will reach their banding density much more quickly in a vertical rotor than in a swinging-bucket rotor. The fixed-angle rotor would occupy an intermediate position. But in a vertical rotor sedimenting particles cannot encounter the wall of the tube in the same way as they do in a fixed-angle rotor. In addition, for tubes of the same volume and dimensions (rotating at the same rpm), the hydrostatic pressure experienced by a particle is much less in a vertical rotor than in a swinging-bucket rotor, since the height of the liquid column is much smaller. The hydrostatic pressure is a function of the square of the height of the liquid column and this pressure has been shown to damage some organelles. In short, for density gradient centrifugation, the vertical rotor has none of the disadvantages of either the swinging-bucket rotor or the fixed-angle rotor.

12. Sample/gradient in vertical rotor The radial thickness of any sample placed on top of a density gradient (A) will also be much smaller in a spinning vertical rotor (B) than in a swinging bucket rotor of the same tube volume. In any separation based on sedimentation velocity, the resolution of the zones, which is dependent on the radial thickness of the sample, may potentially be much better in a vertical rotor than in a swinging-bucket rotor. This factor may outweigh the possible disadvantage of the shorter path length of the vertical rotor. The potential for sample/gradient interfacial instability is also less in a vertical rotor.The radial thickness of any sample placed on top of a density gradient (A) will also be much smaller in a spinning vertical rotor (B) than in a swinging bucket rotor of the same tube volume. In any separation based on sedimentation velocity, the resolution of the zones, which is dependent on the radial thickness of the sample, may potentially be much better in a vertical rotor than in a swinging-bucket rotor. This factor may outweigh the possible disadvantage of the shorter path length of the vertical rotor. The potential for sample/gradient interfacial instability is also less in a vertical rotor.

13. Self-generated gradients Some gradient solutes, whose molecules are sufficiently dense, will sediment, just as any other particle does, if placed in a sufficiently high centrifugal field. As a result of the sedimentation of the molecules, a solute gradient is formed – this is called a self-generated gradient. This phenomenon is well established with heavy metal salts such as CsCl and Cs2SO4 which are used for the banding of DNA and RNA. But some of the newer gradient solutes such as Nycodenz® and iodixanol will do this as well. Because iodixanol has almost double the molecular mass of Nycodenz®, this molecule is particularly suited to the formation of self-generated gradients. The mathematical theory behind the prediction of the shape of self-generated gradients is very complex and cannot be described here. In this very simplistic interpretation of the events, an iodixanol solution is divided into a series of imaginary vertical blocks across which a centrifugal field is established. In the median blocks solute molecules which are lost to the adjacent block on the right are compensated for by molecules arriving from the adjacent block on the left, so the far left and right blocks lose and gain solute molecules respectively. The procedure continues with time during which there is a progressive loss of solute molecules on the left, a progressive build-up on the right and the constant concentration in the middle progressively shrinks. The actual density profile at any time is influenced by the fact that the RCF increases from left to right and that diffusion of the solute in the opposite direction is occurring down the concentration gradient that is formed.Some gradient solutes, whose molecules are sufficiently dense, will sediment, just as any other particle does, if placed in a sufficiently high centrifugal field. As a result of the sedimentation of the molecules, a solute gradient is formed – this is called a self-generated gradient. This phenomenon is well established with heavy metal salts such as CsCl and Cs2SO4 which are used for the banding of DNA and RNA. But some of the newer gradient solutes such as Nycodenz® and iodixanol will do this as well. Because iodixanol has almost double the molecular mass of Nycodenz®, this molecule is particularly suited to the formation of self-generated gradients. The mathematical theory behind the prediction of the shape of self-generated gradients is very complex and cannot be described here. In this very simplistic interpretation of the events, an iodixanol solution is divided into a series of imaginary vertical blocks across which a centrifugal field is established. In the median blocks solute molecules which are lost to the adjacent block on the right are compensated for by molecules arriving from the adjacent block on the left, so the far left and right blocks lose and gain solute molecules respectively. The procedure continues with time during which there is a progressive loss of solute molecules on the left, a progressive build-up on the right and the constant concentration in the middle progressively shrinks. The actual density profile at any time is influenced by the fact that the RCF increases from left to right and that diffusion of the solute in the opposite direction is occurring down the concentration gradient that is formed.

14. Self-generated gradient strategy 1 The use of a self-generated gradient allows the sample to be simply adjusted to a uniform starting concentration of iodixanol; this is used to fill a suitable tube, which is then sealed before being transferred to a vertical rotor. 2 At the running speed the gradient is formed and the particles either move to their buoyant density position. 3 During deceleration from 2000 rpm the gradient reorients; the bands move further apart and become slightly broader during this process. Each gradient is identical and the lack of any interfaces and the dilute nature of the sample at zero time may considerably improve the resolution.1 The use of a self-generated gradient allows the sample to be simply adjusted to a uniform starting concentration of iodixanol; this is used to fill a suitable tube, which is then sealed before being transferred to a vertical rotor. 2 At the running speed the gradient is formed and the particles either move to their buoyant density position. 3 During deceleration from 2000 rpm the gradient reorients; the bands move further apart and become slightly broader during this process. Each gradient is identical and the lack of any interfaces and the dilute nature of the sample at zero time may considerably improve the resolution.

15. Near-vertical rotors are best of all A problem with a vertical rotor is that it is necessary to create a gradient whose (a) highest density is greater than that of the densest particle in the sample and (b) lowest density is less than that of the least dense particle. If this is not the case then the most and least dense particles will reach the wall of the rotor. During the subsequent reorientation of the gradient and during the harvesting of the gradient, this material may contaminate the rest of the gradient. In a near vertical rotor, if this situation occurs, dense pelleted material or light floating material will not create such problems. A problem with a vertical rotor is that it is necessary to create a gradient whose (a) highest density is greater than that of the densest particle in the sample and (b) lowest density is less than that of the least dense particle. If this is not the case then the most and least dense particles will reach the wall of the rotor. During the subsequent reorientation of the gradient and during the harvesting of the gradient, this material may contaminate the rest of the gradient. In a near vertical rotor, if this situation occurs, dense pelleted material or light floating material will not create such problems.

16. Iodixanol self-generated gradient requirements Rotors with a short sedimentation path length ~ 17 mm RCF of 180,000-350,000gav For rapid, efficient formation of all self-generated gradients, a rotor with a relatively short sedimentation path length is required. Only in these rotors can gradients that are more or less linear with volume be created in a reasonably short time < 4h). So vertical rotors are the rotors of choice, although some small volume fixed-angle rotors also fit the bill. Long path length rotors, such as swinging-bucket rotors tend to produce gradients which retain the very shallow middle section even after long periods of centrifugation. Although self-generated gradients will form at RCFs as low as 180,000gav, the optimal formation occurs at approx 350,000gav.For rapid, efficient formation of all self-generated gradients, a rotor with a relatively short sedimentation path length is required. Only in these rotors can gradients that are more or less linear with volume be created in a reasonably short time < 4h). So vertical rotors are the rotors of choice, although some small volume fixed-angle rotors also fit the bill. Long path length rotors, such as swinging-bucket rotors tend to produce gradients which retain the very shallow middle section even after long periods of centrifugation. Although self-generated gradients will form at RCFs as low as 180,000gav, the optimal formation occurs at approx 350,000gav.

17. Effect of time on iodixanol self-generated gradient in the Beckman TLN100 at 365,000g Tubes for the Beckman TLN100 rotor filled with either 15% or 20% iodixanol were centrifuged at 365,000g for either 1 or 3 h and unloaded from the bottom (see Slide 29) into 15 fractions, whose density was determined by refractive index. The typical early S-shaped gradient after 1 h was transformed into a gradient that was more or less linear over about 80% of its volume and slightly more steep in the bottom 20% after 3 h. The starting concentration dictates the density range of the gradient. Tubes for the Beckman TLN100 rotor filled with either 15% or 20% iodixanol were centrifuged at 365,000g for either 1 or 3 h and unloaded from the bottom (see Slide 29) into 15 fractions, whose density was determined by refractive index. The typical early S-shaped gradient after 1 h was transformed into a gradient that was more or less linear over about 80% of its volume and slightly more steep in the bottom 20% after 3 h. The starting concentration dictates the density range of the gradient.

18. Effect of RCF and [iodixanol] in Beckman VTi65.1 (3 h) The progression of the shape of the gradient from S-shaped to more linear is also seen by keeping the time constant and increasing the RCF. Tubes for the Beckman VTi65.1 rotor filled with either 15% or 30% iodixanol were centrifuged for 3 h at either 170,000gav or 353,000gav; then unloaded from the top (see Slides 30-32) into 11 fractions whose density was determined by refractive index. Another factor which influences the shape of the gradient is temperature.The progression of the shape of the gradient from S-shaped to more linear is also seen by keeping the time constant and increasing the RCF. Tubes for the Beckman VTi65.1 rotor filled with either 15% or 30% iodixanol were centrifuged for 3 h at either 170,000gav or 353,000gav; then unloaded from the top (see Slides 30-32) into 11 fractions whose density was determined by refractive index. Another factor which influences the shape of the gradient is temperature.

19. Removal of banded material using a syringe Obvious bands may be harvested from an open-topped tube using a syringeObvious bands may be harvested from an open-topped tube using a syringe

20. Collecting a band from a sealed tube Clearly banded material in an open-topped tube can be simply recovered using a syringe and metal cannula. Banded material in a sealed tube may be harvested by first piercing the tube close to the top with a syringe needle attached to an “open” syringe. With the bezel uppermost, a second needle is inserted just below the band to be harvested. When the plunger of the syringe is withdrawn, air can enter the tube to displace the liquid, through the upper syringe.Clearly banded material in an open-topped tube can be simply recovered using a syringe and metal cannula. Banded material in a sealed tube may be harvested by first piercing the tube close to the top with a syringe needle attached to an “open” syringe. With the bezel uppermost, a second needle is inserted just below the band to be harvested. When the plunger of the syringe is withdrawn, air can enter the tube to displace the liquid, through the upper syringe.

21. Harvest gradient into equal volume fractions by tube puncture There are several devices that may be used to harvest a gradient from a tube into a series of equal volume fractions. Tube puncture may be used for any thin-walled tube. Sealed tubes may be handled this way, if a small bleed hole is made into the top of tube with a syringe needle to allow ingress of air. The Beckman Fraction Recovery System incorporates a very effective tube puncturing device which holds the tube firmly and seals the bottom of the tube. In its simplest form, the effluent from the hollow needle used to puncture the tube is collected by gravity in a series of tubes. This manual form of collection is very tedious and the flow rate varies greatly with the viscosity of the gradient. The resolution of the gradient is however best maintained by this simple method. In the more elaborate form shown here the effluent flow is made more regular by incorporating a small peristaltic pump and the gradient is collected in a multi-well plate. If the internal diameter of the tubing is kept as small as possible there is little loss of resolution. Collection of equal volume fractions is not easy either manually or using a pump and fraction collector. The drop size of the effluent gradient varies with the viscosity of the liquid, so either manual or automatic drop counting will give a variable fraction volume. Automatic collection on a time basis may therefore be the mode of choice with a fraction collector, but loss of material between fractions is more likely (even if the pump operation is transiently interrupted at each fraction change). In the manual mode the gradient might be collected in tubes with a calibration mark as a guide. There are several devices that may be used to harvest a gradient from a tube into a series of equal volume fractions. Tube puncture may be used for any thin-walled tube. Sealed tubes may be handled this way, if a small bleed hole is made into the top of tube with a syringe needle to allow ingress of air. The Beckman Fraction Recovery System incorporates a very effective tube puncturing device which holds the tube firmly and seals the bottom of the tube. In its simplest form, the effluent from the hollow needle used to puncture the tube is collected by gravity in a series of tubes. This manual form of collection is very tedious and the flow rate varies greatly with the viscosity of the gradient. The resolution of the gradient is however best maintained by this simple method. In the more elaborate form shown here the effluent flow is made more regular by incorporating a small peristaltic pump and the gradient is collected in a multi-well plate. If the internal diameter of the tubing is kept as small as possible there is little loss of resolution. Collection of equal volume fractions is not easy either manually or using a pump and fraction collector. The drop size of the effluent gradient varies with the viscosity of the liquid, so either manual or automatic drop counting will give a variable fraction volume. Automatic collection on a time basis may therefore be the mode of choice with a fraction collector, but loss of material between fractions is more likely (even if the pump operation is transiently interrupted at each fraction change). In the manual mode the gradient might be collected in tubes with a calibration mark as a guide.

22. Upward displacement In upward displacement, a dense solution (Perfluorodecalin – an inert low viscosity fluorocarbon with a density of approx.1.9 g/ml is recommended) is introduced to the bottom of the tube and the gradient harvested low density end first using a simple conical collection device (fashioned from a block of acrylic) on top of the tube. The Perfluordecalin may be introduced from a burette and pump via a tube inserted through the collection head.This method can be used with either thin-walled or thick-walled tubes. The Beckman Fraction Recovery system also includes a device for collection from the top of the tube. Alternatively (for thin-walled tubes only) the Perfluorodecalin may be directed into the bottom of the centrifuge tube via the hollow needle of a tube puncture device. The burette allows easy collection of equal volume fractions and this is the only guaranteed and accurate way of accomplishing this task.In upward displacement, a dense solution (Perfluorodecalin – an inert low viscosity fluorocarbon with a density of approx.1.9 g/ml is recommended) is introduced to the bottom of the tube and the gradient harvested low density end first using a simple conical collection device (fashioned from a block of acrylic) on top of the tube. The Perfluordecalin may be introduced from a burette and pump via a tube inserted through the collection head.This method can be used with either thin-walled or thick-walled tubes. The Beckman Fraction Recovery system also includes a device for collection from the top of the tube. Alternatively (for thin-walled tubes only) the Perfluorodecalin may be directed into the bottom of the centrifuge tube via the hollow needle of a tube puncture device. The burette allows easy collection of equal volume fractions and this is the only guaranteed and accurate way of accomplishing this task.

23. Labconco Auto Densi-flow collects from meniscus 1 Essentially the Labconco Auto Densi-flow comprises a collection head [C] at the end of a narrow metal tube which is connected (via flexible silicone tubing) to a peristaltic pump [P]. An electronic probe [EP] adjacent to C is connected to the power supply of a motor [M]. EP and C are mounted in a block [B] linked to the motor drive. 2 When the motor is activated, the block descends until the tip of the probe hits the meniscus of the gradient. The change in conductivity registered by the probe deactivates the motor. 3 When the pump is switched on, the gradient is aspirated through the collection head; the meniscus thus falls so the probe is no longer in the electrically-conducting liquid. 4 The motor is reactivated until the probe re-enters the meniscus and more liquid is aspirated….. etc. For illustrative purposes the procedure appears to occur in a series of steps, but in practise the downward movement of the block and the aspiration of the liquid through the pump are continuous. It is widely regarded as one of the best methods of gradient collection and can be used for gradient volumes of 3 ml and above. The pump and motor of the machine can be used in the reverse direction to deposit a gradient in a tube from a two-chamber gradient maker (see Slide 6) dense-end first. In this way C always lies at the meniscus of the ascending liquid in the tube.1 Essentially the Labconco Auto Densi-flow comprises a collection head [C] at the end of a narrow metal tube which is connected (via flexible silicone tubing) to a peristaltic pump [P]. An electronic probe [EP] adjacent to C is connected to the power supply of a motor [M]. EP and C are mounted in a block [B] linked to the motor drive. 2 When the motor is activated, the block descends until the tip of the probe hits the meniscus of the gradient. The change in conductivity registered by the probe deactivates the motor. 3 When the pump is switched on, the gradient is aspirated through the collection head; the meniscus thus falls so the probe is no longer in the electrically-conducting liquid. 4 The motor is reactivated until the probe re-enters the meniscus and more liquid is aspirated….. etc. For illustrative purposes the procedure appears to occur in a series of steps, but in practise the downward movement of the block and the aspiration of the liquid through the pump are continuous. It is widely regarded as one of the best methods of gradient collection and can be used for gradient volumes of 3 ml and above. The pump and motor of the machine can be used in the reverse direction to deposit a gradient in a tube from a two-chamber gradient maker (see Slide 6) dense-end first. In this way C always lies at the meniscus of the ascending liquid in the tube.

24. Labconco Auto Densi-flow linked to a Gilson FC205 Fraction Collector The ultimate sophistication in gradient collection – a Labconco Auto Dens-flow linked to a Gilson Fraction Collector; the gradient from a 38 ml tube being collected in a Greiner Bio-One Master Block. The Auto-Densi-Flow has been used for Beckman Optiseal tubes as small as 3 ml.The ultimate sophistication in gradient collection – a Labconco Auto Dens-flow linked to a Gilson Fraction Collector; the gradient from a 38 ml tube being collected in a Greiner Bio-One Master Block. The Auto-Densi-Flow has been used for Beckman Optiseal tubes as small as 3 ml.